§1.1 Field of the Invention
The present invention concerns nanogels and producing nanogels in general. In particular, the present invention concerns nanogel production using liposomes as reactors.
§1.2 Related Art
Hydrogels are networks of hydrophilic polymers that can be swollen with water. They exhibit both liquid properties (because the major constituent is water) and solid properties (because of crosslinking during polymerization). Further, depending on monomers incorporated into the network, the system becomes sensitive to environmental conditions such as pH, temperature, ion concentration, electric and magnetic fields, light and solvent. This sensitivity is evidenced in the polymer network's capacity to swell or shrink in response to changes in its environment.
Artificial systems of spherical hydrogel particles have already found a variety of biomedical applications in drug delivery, drug targeting, protein separation, enzyme immobilization, etc.
It has been reported that some polymer gels can swell or shrink discontinuously and reversibly in response to many different stimuli (temperature, pH, ions, electric fields or light) depending on the chemical composition of the gel/solvent system. The volume change can be as large as a thousand-fold. Unfortunately, however, macroscopic gels respond to the environmental changes on a rather long time scale. The article Tanaka et al., J. Chem. Phys., 90: 5161 (1989) (This article is incorporated herein by reference.) showed that for a spherical gel, the time required for swelling or shrinking is inversely proportional to the square of its radius. Therefore, decreasing the size of a spherical hydrogel should reduce the time needed for shrinking and/or swelling. A decrease in the hydrogel size to nanometer scale, and the expected quick swell and/or shrink times of such nanometer scale hydrogels should greatly widen potential applications for hydrogels.
Work related to preparing and characterizing submicrometer-scale hydrogel particles has intensified recently (See, e.g., the articles: R. Pelton, Adv. Coll. Interface Sci., 85: 1-33 and the references therein (2000); T. Tanaka et al., Phys. Rev. Lett., 45: 1636 (1980); G. M. Eichenbaum et al., Macromolecules, 31: 5084 (1998); G. M. Eichenbaum et al., Macromolecules, 32: 4867 (1999); G. M. Eichenbaum et al., Macromolecules, 32: 8996 (1999); P. Markland et al., J. Biomed. Mater. Res., 47: 595 (1999); C. Wu, Polymer, 39: 4609 (1998); K. Kratz et al., Polymer, 42: 6631 (2001); M. Monshipouri et al., U.S. Pat. No. 5,626,870 (1997), hereafter referred to as “the Monshipouri Patent”; V. P. Torchilin et al., Macromol. Chem., Rapid Commun. 8: 457 (1987), hereafter referred to as “the Torchilin article”; K. Gao et al., Biochim. Biophys. Acta, 897: 337 (1987); T. Jin et al., FEBS Lett., 397: 70 (1996); P. F. Kiser et al., Nature, 394: 459 (1998). These articles and the patent are incorporated herein by reference). However, in all the referenced works (except the Monshipouri Patent and the Torchilin article) the sizes of hydrogel particles varied on the micrometer scale. (In these works, optical or electron microscopy was used for characterization.)
Liposomes, or lipid vesicles, are phospholipid assemblies of a flexible, cell membrane-like lipid bilayer, the surface of which acts as a permeability barrier. Liposomes may differ in size (small or large) and lamellarity (multilamellar or unilamellar), resulting in liposomal suspensions of multilamellar vesicles (MLV), large unilamellar vesicles (LUV), or small unilamellar vesicles (SUV). Liposomes have completely closed lipid bilayer membranes containing an entrapped aqueous volume. This structure provides the conditions for using the liposomal interior as a reaction vessel for carrying out chemical or biochemical reactions since different compounds may be trapped in the liposome. For example, liposomes may be employed as reactors for nanometer-scale reactions such as hydrogel production.
The size of hydrogels produced in liposomes is dependent on the sizes of the liposomes themselves. U.S. Pat. No. 5,626,870 (hereafter “the Monshipouri patent”) describes hydrogels with sizes ranging from 50-3000 nm. The Torchilin article describes hydrogel-containing liposomes with average diameter of about 650 nm. Liposomes used to produce hydrogels with this range of diameters may be multilamellar (d>1000 nm) or unilamellar (LUV: 100 nm <d<1000 nm; or SUV: d<100 nm). Production of each class of liposomes required different methods of preparation, such as hydrodynamic shear (U.S. Pat. No. 5,082,664), reverse phase evaporation (U.S. Pat. No. 4,235,871), sonication (U.S. Pat. No. 4,089,801), detergent dialysis (Proc. Nat. Acad. Sci. USA, 1979, 145-149), and extrusion through membrane pores (U.S. Pat. No. 5,008,050). These methods, however, make it difficult to control liposome size on a large scale and often result in a loss of material, causing low liposome yields.
The Monshipouri patent prepared LUV liposomes with diameters of about 800 nm using extrusion through 800-nm membranes. Using the extrusion technique, only LUV can be prepared, material can be lost, and it is highly time- and labor-consuming.
The Torchilin article describes preparing LUV liposomes with average diameters of about 650 nm using the reverse phase evaporation method. Using this technique, it is difficult to control liposome size and polydispersity, which is why the authors presented only the average sizes of the particles detected by dynamic light scattering. Moreover, gel containing liposomes and gel particles were not distinguished by scanning electron microscopy.
Centrifugation, filtration and column chromatography have been used to prevent polymerization outside the liposomes, and to remove extra-liposomal initiator (such as calcium ions in the Monshipouri patent) or to separate the monomer-containing liposomes from the extra-liposomal non-entrapped monomer (the Torchilin article). However, these methods result in a loss of phospholipid, and thus have low yields of unilamellar liposomes.
After polymerization, detergent/phospholipid mixed micelles may be removed by centrifugation, as in the Monshipouri patent. A drawback to this technique is that a portion of hydrogel particles can be lost during centrifugation. In the Torchilin article, the detergent and phospholipid were not removed at all.
Furthermore, the Monshipouri patent describes a limited number of hydrogel-forming substances including sodium alginate, chitosan, and K-carrageenan and uses physical polymerization, which requires an initiator of gelation for which a liposomal bilayer is permeable. However, removing these initiators by centrifugation, filtration, or column chromatography results in loss of phospholipid and consequently low yields of unilamellar liposomes. The Torchilin article describes one composition of a hydrogel-forming medium. The presence of initiator (4,4′-azobis(4-cyanovaleric acid)) and accelerator (N,N,N′,N′-tetramethylenediamine, TEMED) indicates the use of redox polymerization, which can proceed without UV-irradiation. There are no compositional details and parameters of polymerization. Moreover, the Torchilin article uses a gel permeation chromatography to prevent polymerization outside the liposomes, which results in loss of monomer-loaded liposomes. Also, the Monshipouri patent and the Torchilin article deal only with aqueous suspensions of hydrogel particles and does not consider producing dry hydrogel particles or delivering water-insoluble compounds into the liposomal reactor.
In view of the limits of the state of the art, hydrogel nanoparticles (nanogels), and methods for their production, are needed.